Breast cancer preferentially metastasizes to the bone, where the five-year relative survival rate falls from 90% to <10%. Although the precise mechanism underlying preferential metastasis is unknown, the bone likely provides a hospitable environment that both attracts breast cancer cells and allows them to colonize and grow. Clinical evidence suggests that breast cancer cells can exist in a dormant state in the bone for up to 25 years, evading immune surveillance and chemotherapeutic treatments which target cycling cells. As a result, breast cancer patients successfully treated for primary breast cancer decades before develop overt macrometastases without prior symptoms. Thus, there is a critical need for elucidating molecular mechanisms facilitating breast cancer dormancy and reactivation. This proposal aims to understand the role of the bone microenvironment in breast cancer cell bone engraftment (Aim 1), dormancy (Aim 2), and reactivation (Aim 3), with the long-term goal of identifying key interactions for therapeutic intervention between the bone microenvironment, bone stromal cells, and breast cancer cells that contribute to breast cancer cell bone dormancy and metastasis. To address these challenges, first, in Aim 1 (mentored), I will determine the importance of bone stromal cells and their secreted factors for metastatic breast cancer cell bone engraftment and colonization. In this aim, I will utilize in-vitro cell culure and novel compartmentalized bioreactor and in-vivo extramedullary bone models that mimic human breast cancer metastasis in-vivo to determine if 1) breast cancer cells form gap junctions with bone stromal cells, 2) alterations in mesenchymal stem cell-derived factors regulate breast cancer cell engraftment, or 3) if formation of cancer-associated fibroblasts is modulated by osteoblast-derived cytokines. In the Second Aim (Independent), I will identify key interactions between bone stromal cells and breast cancer cells that lead to breast cancer cell dormancy in the bone. I will 1) use bone-seeking breast cancer cells and the spontaneous mouse model of breast cancer bone metastasis to identify gap junctions critical to breast cancer cell quiescence. Next, I will 2) determine how crosstalk between osteoblasts and cancer-associated fibroblasts ultimately affects their functions. Finally, I will 3) use cell culture to determine if cancer-associated fibroblasts and osteoblasts express exosomes that impact breast cancer cell dormancy. Lastly, in the Third Aim (Independent), I will uncover mechanisms involving bone stromal cells that contribute towards the reactivation of breast cancer cells and formation of a metastatic niche in bone. Here, I will use 1) short-hairpin RNAs to identify specific osteoblast-derived cytokines that reactivate breast cancer cells utilizing both co-culture and an intratibial mouse model. Additionally, I will use the compartmentalized bioreactor to determine if cancer-associated fibroblasts regulate 2) the differentiation or 3) mediate apoptosis of osteoblasts. Finally, I will 4) determine the importance of cancer-associated fibroblasts in the formation of osteoclasts.